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Pyruvic oxidase, and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof

A technology of pyruvate oxidase and recombinant host cells, which is applied in the field of oxidase, can solve the problems of poor heat resistance, and achieve the effects of good heat resistance, high residual enzyme activity and high stability

Active Publication Date: 2010-03-03
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] An object of the present invention is to overcome the shortcomings of the existing pyruvate oxidase, which has poor heat resistance and can only maintain high stability and activity in a narrow pH range, and provide a kind of pyruvate oxidase that has good heat resistance and can be used in a wide range of pH values. It maintains high stability and activity in the pH range, and has pyruvate oxidase with high residual enzyme activity after repeated freezing and thawing

Method used

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  • Pyruvic oxidase, and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof
  • Pyruvic oxidase, and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof
  • Pyruvic oxidase, and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof

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preparation Embodiment 1

[0060] (1) Synthesis of existing pyruvate oxidase poxB gene

[0061] According to the document ["Characterization and Functional Analysis of the poxB Gene, Which Encodes Pyruvate Oxidase in Lactobacillus plantarum]", Frédérique Lorquet et al., Journal of Bacteriology (Journal of Bacteriology), Vol. 186, No. 12, pp. 3749-3759, June 2004] reported the sequence of pyruvate oxidase from Lactobacillus plantarum, entrusted Jerry Bioengineering (Shanghai) Co., Ltd. to use DNA The synthesizer synthesized DNA fragments, and then connected the obtained DNA fragments with DNA ligation technology, and finally sequenced the obtained DNA fragments with the 310 type automatic DNA sequencer of Applied Biosystems. The sequencing results showed that the obtained DNA fragments contained 1812bp in length. The protein coding gene (starting at the start codon atg and ending at the stop codon taa) is consistent with the above-mentioned literature report, and is the nucleotide sequence shown in SEQ I...

preparation Embodiment 2

[0120] The whole gene synthesis is carried out according to the sequence shown in SEQ ID NO: 3 to obtain the synonymous mutant nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 2. Then, the obtained nucleotide sequence was ligated into pET28a(+) empty vector according to the method of Preparation Example 1, and transformed into Escherichia coli BL21(DE3). The resulting recombinant host cells were cultured under the same conditions, and as a result, the yield of pyruvate oxidase in this example was 149.7% higher than that in Example 1.

preparation Embodiment 3

[0122] The nucleotide sequence shown in SEQ ID NO: 2 was ligated into pET32a(+) empty vector according to the method of Preparation Example 1, and transformed into Escherichia coli BL21(DE3). The resulting recombinant host cells were cultured under the same conditions, and the resulting pyruvate oxidase had a Km value of 5.4×10 -4 mol / L, the output of its pyruvate oxidase is basically equal to that of Preparation Example 1.

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Abstract

The invention discloses a pyruvic oxidase, the amino acid sequence of which is (1) shown in SEQ ID NO: 1, or (2) obtained by losing, adding and / or substituting of one or a plurality of amino acids ofthe amino acid sequence in (1), but the pyruvic oxidase function of which is invariable. The invention also discloses a nucleotide sequence encoding the pyruvic oxidase, and comprises the recombinantvector of the nucleotide sequence, the recombinant host cell of the recombinant vector, and the kit of at least one of the pyruvic oxidase, the nucleotide sequence, the recombinant vector and the recombinant host cell. The pyruvic oxidase has good heat resistance, high activity of residual enzyme after repeated freeze thawing and high stability within wide pH value range.

Description

technical field [0001] The present invention relates to an oxidase, a nucleotide sequence encoding the enzyme, a recombinant vector comprising the nucleotide sequence, a recombinant host cell comprising the recombinant vector, and the oxidase, nucleotide sequence, recombinant A kit of at least one of a vector and a recombinant host cell. More specifically, the present invention relates to a pyruvate oxidase, a nucleotide sequence encoding the pyruvate oxidase, a recombinant vector comprising the nucleotide sequence, a recombinant host cell comprising the recombinant vector, and a recombinant vector comprising the above-mentioned A kit of at least one of pyruvate oxidase, nucleotide sequence, recombinant vector and recombinant host cell. Background technique [0002] Pyruvate oxidase (Pyruvate oxidase, EC1.2.3.3) is a widely used enzyme preparation for clinical diagnosis. It is mainly used for the activity determination of blood alanine aminotransferase (ALT) and / or asparta...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N1/21C12N1/19C12N5/10C12Q1/52C12R1/19
Inventor (请求不公开姓名)
Owner BEIJING LEADMAN BIOCHEM
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